Composition containing fine particles for supporting biologically active substance thereon or having the same supported thereon and method for preparing these

ABSTRACT

Provided are a dispersion comprising organic-inorganic hybrid type particles carried thereon with a biologically active substance, wherein the above particles can be obtained by allowing a block copolymer represented by Formula (I):  
     PEG-block-poly(carbo)  (I)  
     (wherein PEG represents a polyethylene glycol segment, and carbo represents a repetitive unit having a carboxylate ion on a side chain) and an aqueous medium system capable of forming hydroxyapatite to coexist with the biologically active substance, and a preparing method for the same.

TECHNICAL FIELD

[0001] The present invention relates to a composition containingorganic-inorganic hybrid particles for carrying a biologically activesubstance or carried thereon with it and a method for preparing them.The above biologically active substance can preferably be a highmolecular polyvalent anionically chargeable compound (for example, poly-or oligonucleotide and poly- or oligopeptide).

BACKGROUND ART

[0002] A crystal of calcium phosphate (hydroxyapatite) formed when acalcium aqueous solution is mixed with a phosphoric acid aqueoussolution so that a supersaturation state is obtained has a property tobond with DNA. A method in which calcium phosphate and DNA arecoprecipitated making use of this property has been widely utilized as amethod for introducing DNA into a cell. The problems of this methodinclude a very narrow range of an optimum condition, which makeshandling thereof difficult, and less liability to obtainreproducibility. In particular, growth in the crystal of calciumphosphate is very fast to form the giant crystal, and therefore it ispointed out that an efficiency of introducing DNA is reduced if acalcium aqueous solution and a phosphoric acid aqueous solution are notquickly worked on a cell after mixing. Also, hydroxyapatite is used as abase substance (for example, a carrier for chromatography) for adsorbinga biologically active substance including polynucleotide such as DNA,other peptides or polypeptide.

[0003] In the foregoing method for introducing DNA (or gene) into acell, development of a method for controlling a growth in a crystal ofcalcium phosphate and a particle diameter thereof is considered to beimportant to a rise in an introducing efficiency of DNA, areproducibility and a storage stability. Further, if substances carriedor adsorbed on such crystal can widely be used as various biologicallyactive substances without being restricted to DNA described above and aparticle diameter of a particle formed can be controlled, it isconsidered to be important in providing a carrier system which canwidely be used for carrying medicines or a delivery system formedicines. Accordingly, an object of the present invention is to providea composition useful for forming particles which not only enhance anefficiency of introducing DNA into a cell but also have a wide andcontrolled particle diameter and which can stably carry a biologicallyactive substance and conveniently deliver the biologically activesubstance to a target cell, a desired tissue or a local site.

DISCLOSURE OF THE INVENTION

[0004] The present inventors have continued researches in order tocontrol a growth in a crystal of calcium phosphate (mainlyhydroxyapatite) in an aqueous solution containing a calcium ion and aphosphoric acid ion and a particle diameter thereof. As a resultthereof, they have found that calcium phosphate particles into which DNAor the other biologically active substances is introduced or in whichthey coexist can be formed while controlling a particle diameter thereofwhen a calcium ion is reacted with a phosphoric acid ion under thecoexistence of DNA or the other biologically active substances in anaqueous solution in which present is a specific block copolymercontaining a hydrophilic and nonionic polyethylene glycol (PEG) segmentand a polyanionic segment originating in a carboxyl group. In additionthereto, it has been confirmed that a particle diameter of suchparticles can be controlled, if necessary, to a submicron order (several100 nm) or less and that an aqueous dispersion system containing suchparticles can stably be stored under an ambient condition withoutproducing precipitates. Further, it has been found as well that suchaqueous dispersion system can be turned into a composition of a driedtype (for example, freeze-dried) and that it can be then reconstitutedto the same aqueous dispersion system.

[0005] Hence, according to the present invention, provided is an aqueouscomposition for forming (or used for forming) organic-inorganic hybridtype particles carried thereon with a biologically active substance,wherein the particles described above comprise a block copolymer havinga structure represented by Formula (I):

PEG-block-poly(carbo)  (I)

[0006] (wherein PEG represents a polyethylene glycol segment, and carborepresents a repetitive unit having a carboxylate ion on a side chain),a calcium ion (Ca⁺²) and a phosphoric acid ion (PO₄ ³⁻) as essentialcomponents.

[0007] Further, provided as another embodiment of the present inventionis a composition comprising organic-inorganic hybrid type particlescarried thereon with a biologically active substance, wherein theparticles described above are formed from a block copolymer having astructure represented by Formula (I):

PEG-block-poly(carbo)  (I)

[0008] (wherein PEG represents a polyethylene glycol segment, and carborepresents a repetitive unit having a carboxylate ion at a side chain),a calcium ion (Ca⁺²), a phosphoric acid ion (PO₄ ³⁻) and the abovebiologically active substance, and the above particles have an averageparticle diameter of 50 to 60 nm.

[0009] Further, provided are an aqueous dispersion composition forforming organic-inorganic hybrid type particles carried thereon withsuch biologically active substance, a method for preparing a compositioncontaining the above particles carried thereon with a biologicallyactive substance and a method for indroducing the above biologicallyactive substance, particularly poly-or oligonucleotide into a cell,comprising a step of incubating the above composition under thecoexistence of a cultured cell or injecting it into a suitable part ofan animal.

[0010] It has so far been known that when a phosphoric acid ion is addedto a coagulating solution of modified PEO-block-PMAA (PEO is apolyethylene oxide or polyethylene glycol segment; PMAA is apolymethacrylic acid segment; and among PMAA, three segments aremodified with C₁₂alkane) after adding CaCl₂, a hybrid type structure ofhydroxyapatite/PEG-block-PMAA-C₁₂ having a neuronal structure isobtained (Henbnur Coelfer, Macromol. Rapid Commun. 2001, 22, 219 to252).

[0011] According to the present invention, provided are particles whichare substantially spherical regardless of that a biologically activesubstance is further carried or absent and a means capable of forminguniform particles which have a particle diameter suited for beingefficiently introduced into a cell by endocytosis and in which aparticle diameter has a narrow distribution. Particles in which abiologically active substance is absent shall be useful as ahydroxyapatite material of a new form comprising fine particles.Further, a system containing particles carried thereon with abiologically active substance shall deliver the biologically activesubstance to a target and expand a range of a usefulness in the abovesubstance.

BRIEF DESCRIPTION Of The DRAWINGS

[0012]FIG. 1 is a graph showing an influence of PAA and PEG-PAA exertedto a growth in crystal of CaP, wherein (a) is a graph showing a changein a transmittance with the passage of time in the presence of PAA (PAAconcentration: (□) 14 μg/mL, (⋄) 14 μg/mL, (◯) 43 μg/mL and (Δ) 57μg/mL), and (b) is a transmittance after 3 minutes since mixing thesolutions ((□) PAA, (◯) PEG/PAA (blended) and (Δ) PEG-PAA).

[0013]FIG. 2 is a graph showing a result of measuring a particlediameter of CaP particles by dynamic light scattering.

[0014]FIG. 3 is a graph showing a change in a polydispersity of CaPparticles to a PEG-PAA concentration.

[0015]FIG. 4 is a graph showing the same measuring result as in FIGS. 2and 3 regarding particles prepared without adding DNA in preparing theparticles.

[0016]FIG. 5 is an elution pattern showing a result of determining DNAincluded in CaP particles by HPLC; (a) upper line: only DNA and (b) agraph showing a change in an amount of included DNA to a PEG-PAAconcentration ((◯): eluate 1 and (Δ): eluate 2).

[0017] In FIG. 6, A) is a microscopic image in place of a drawingshowing the state of a cell after working only DNA on the cell. In thiscase, granular fluorescence indicating introduction by endocytosis isobserved.

[0018] B) is a microscopic image in place of a drawing showing the stateof a cell after working a precipitate (PEG-PAA 70 μg/ml) of CaP on thecell. In this case, an image showing that the precipitate is adsorbed onthe surface of the cell is obtained.

[0019] In FIG. 7, C) is a microscopic image in place of a drawingshowing the state of a cell after working CaP particles (PEG-PAA 280μg/ml) (in the non-coexistence of PMA) on the cell. In this case,granular fluorescence indicating introduction of the CaP particles byendocytosis is observed. The same result was obtained in a PEG-PAA of140 μg/ml.

[0020] D) is a microscopic image in place of a drawing showing the stateof a cell after working CaP particles (PEG-PAA 280 μg/ml) (in thecoexistence of PMA) on the cell. In this case, observed is a fluorescentimage showing localization of the CaP particles in a nucleus as well asgranular fluorescence indicating that the CaP particles are introducedby endocytosis.

[0021] In FIG. 8, E) is a microscopic image in place of a drawingshowing the state of a cell after working only a fluorescent moleculeRhodamine on the cell. In this case, fluorescence is observed all over acytoplasma, and it is shown that a fluorescent molecule does notselectively move to a nucleus.

[0022]FIG. 9(a) is a graph showing that a particle diameter of theorganic-inorganic hybrid particles according to the present inventionchanges by a change in a copolymer concentration and a phosphoric acidconcentration in Example 9, and (b) is a graph showing a polydispersityof the respective particles.

[0023]FIG. 10 is a graph showing a dependency of a DNA incorporatedamount in the particles on copolymer and phosphoric acid concentrations.

[0024]FIG. 11(a), (b) and (c) are graphs showing results obtained byevaluating introduction of DNA into a cell in Example 8.

[0025]FIG. 12 is a graph showing a result of a test carried out in orderto evaluate a toxicity of the particles prepared in Example 9.

[0026]FIGS. 13A and B are graphs showing results obtained byinvestigating an expression activity of a plasmid DNA-incorporatingparticle in a cell in Example 10.

BEST MODE FOR CARRYING OUT THE INVENTION

[0027] According to the present invention, a block copolymer having astructure represented by Formula (I):

PEG-block-poly(carbo)  (I)

[0028] (wherein PEG represents a polyethylene glycol segment, and carborepresents a repetitive unit having a carboxylate ion on a side chain)is characterized by being used in common in a system forming calciumphosphate (hydroxyapatite).

[0029] As described above, when referred to as “calcium phosphate” or“hydroxyapatite” in the present specification, it means a mixture ofsalts which contains mainly calcium phosphate (Ca₃(PO₄)₂) andhydroxyapatite (Ca₁₀(OH)₂(PO₄)₆) and which is formed from a calciumcation (Ca⁺²) and a phosphoric acid anion (PO₄ ³⁻) in an aqueoussolution. It is intended that 50% by weight or more of a salt of ahydroxyapatite type is preferably contained therein.

[0030] Poly(carbo) which is one segment in the block copolymer describedabove represents a polymer segment comprising a repetitive unit having acarboxylate ion at a side chain, and the kind of a starting materialproviding such repetitive unit does not matter as long as it meets theobjects of the present invention. However, capable of being preferablygiven is a repetitive unit originating in a compound having at least onecarboxyl group selected from the group consisting of aspartic acid,glutamic acid, methacrylic acid, acrylic acid and N-acetylhyalobiuronicacid (a repetitive unit of hyaluronic acid). In such Poly(carbo), afixed carboxyl group can stay in the form of an ester (for example,lower alkyl having up to 6 carbon atoms or benzyl ester) according to aproduction method of the block copolymer described above. According tothe present invention, a residue in the ester of such form may becontained in an amount of up to about 50%, preferably less than 10% andparticularly preferably 0% as long as introduction into or adsorptiononto calcium phosphate (or hydroxyapatite) is not hindered.

[0031] The term “having the structure” represented by Formula (I)intends that a linkage group between PEG and poly(carbo) and an end ofPEG or poly(carbo) can have any group or part as long as it meets theobjects of the present invention.

[0032] A copolymer represented by any one of the following Formulas(II-a), (II-b), (III-a) and (III-b) can be given as the block copolymerparticularly preferably used in the present invention:

[0033] In the respective formulas, the respective codes each haveindependent meanings;

[0034] A represents a hydrogen atom or a substituted or unsubstitutedalkyl group having up to 12 carbon atoms;

[0035] L represents a single bond, NH, CO or X(CH₂)_(p)Y, in which Xrepresents OCO, OCONH, NHCO, NHCOO, NHCONH, CONH or COO; Y represents NHor CO; and p represents an integer of 1 to 6;

[0036] T represents a hydrogen atom, a hydroxyl group or -ZR, in which Zrepresents a single bond, CO, O or NH, and R represents a substituted orunsubstituted hydrocarbon group having up to 12 carbon atoms;

[0037] m represents an integer of 4 to 2500; and

[0038] x+y or z represents an integer of 5 to 300, provided that acarboxylate ion present can form a carboxyester residue in an amount ofup to 50%. Also, the mark “·”between an α-aspartic acid unit and aβ-aspartic acid unit in Formulas (II-a) and (II-b) described above meansthat these units are present at random.

[0039] The block copolymer in which m is an integer of 12 to 2500 in theformulas described above and in which x+y or z is an integer of 5 to 50can more preferably be used.

[0040] The definitions of the respective groups and the respective partsin the formulas described above have, to be specific, the followingmeanings. The “alkyl group having up to 12 carbon atoms (hereinafterabbreviated as C₁₂, and such describing manner shall be applied as wellwhen representing the other groups having carbon atoms)” is an alkylgroup which may be linear or branched and represents, for example,methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, n-pentyl,n-hexyl and n- or iso-dodecyl. A substituent for such alkyl groups maybe any group as long as it meets the objects of the present invention,and capable of being preferably given are a hydroxyl group, a carboxylgroup, a group represented by a formula R¹R²CH— (wherein R¹ and R²represent independently C₁₋₁₀ alkyloxy, aryloxy or aryl-C₁₋₃ alkyloxy orrepresent ethylenedioxy (—O—CH(R′)—CH—O—, wherein R′ is a hydrogen atomor a C₁₋₆ alkyl group) which may be substituted with C₁₋₁₀ alkyl) and agroup represented by a formula R¹′ R²′ NCH₂— (wherein R¹′ and R²′represent independently an amino protective group of an organic silyltype, for example, a trialkylsilyl group, or R¹′ and R²′ represent anatomic group which can form a 4- to 7-membered disilaneazacycloheterocyclic ring together with a nitrogen atom to which they arebonded). For example, the group represented by the formula R¹R² CH—represents so-called acetal or ketal part and can readily be convertedto OCH— (aldehyde group) by moderate hydrolysis. On the other hand, thegroup represented by the formula R¹′ R²′ NCH₂-can readily be convertedto H₂N— in a solution containing, for example, tetraalkylammoniumchloride. Accordingly, the block copolymer represented by Formula(II-a), (II-b), (III-a) or (III-b) having such substituent is used toform the organic-inorganic hybrid type particles (for example, polymermicelle) according to the present invention, and then the foregoingsubstituent which is usually present on the shell or the surface of theabove particles is converted to an aldehyde group or an amino group; andpolypeptide showing a specific bonding property in, for example, anantibody is advantageously subjected to covalent bonding with the aboveparticles via the functional group thus obtained. Thus, the aboveparticles can be provided with a target directivity. A method forgetting a PEG segment having such substituent is known and can bereferred to WO96/33233 (or corresponding U.S. Pat. No. 5,925,720) in thecase of, for example, the R¹R²CH— group.

[0041] The block copolymer described above can be selected fromconventionally known ones. It can be produced by forming first apolyethylene glycol segment and then forming a poly(amino acid) segmentaccording to, for example, a method described in Japanese PatentApplication Laid-Open No. 107565/1994 or by forming first a polyethyleneglycol segment and then forming a poly((meth)acrylic acid) segmentaccording to a method described in WO97/06202. As another method, aconventionally known linkage group may be present, if necessary, betweena PEG segment and a poly(carbo) segment. However, the copolymerdescribed above shall not be restricted to those obtained by theseproduction methods, and copolymers which can be obtained by producing inadvance independently polymers constituting both segments and thencombining them by a suitable method can be used as well in the presentinvention.

[0042] In the block copolymer thus obtained, T in Formulas (II-a),(II-b), (III-a) and (III-b) described above or an end groupcorresponding thereto is usually a hydrogen atom or a hydroxyl group,and a -ZR group can be introduced into these end groups by aconventionally known method. Such R shall not be restricted, andhydrocarbon groups such as —CH₃, —CH₂CH₃, —CH═CH₂,

[0043] can be given. Such groups can be introduced according to themethod described in U.S. Pat. No. 5,925,720 described above.

[0044] The biologically active substance which is intended to be or iscarried on (or included in) the organic-inorganic hybrid type particlesaccording to the present invention may be any biologically activesubstance, though shall not theoretically restricted, as long as it isan organic compound which can be included in or adsorbed on a complex ora cross-linked matrix formed by a block copolymer (particularly acarboxylate ion) and a calcium ion or hydroxyapatite. Preferably,however, it may be any biologically active substance selected from thegroup consisting of poly- or oligonucleotide (used in a conceptincluding DNA or RNA or peptide derivatives thereof) and poly oroligopeptide or derivatives thereof. Such DNA can be a conventionallyknown cancer-inhibiting gene and others, a gene necessary formaintaining a homemostasis of organisms and an anti-sense of the otherspecific genes. According to the present invention, these genes canefficiently be introduced into a target cell. Further, in order to usein combination with the organic-inorganic hybrid type particles carriedthereon with such genes or independently, polypeptide (including singleprotein and protein having a sugar chain) which is a proliferativefactor known to accelerate a differentiation and inhibit a proliferationin a cell and induce apotosis can be selected as a biologically activesubstance carried on the above particles. For example, TGF-β₁, TGF-β₂,TGF-α, connective tissue-activating peptide, a tumor necrotizing factor,an insulin-like proliferative factor, interleukin, a colony stimulatingfactor and a nerve proliferating factor can be given as thesebiologically active substances.

[0045] The factor effective for healing of wound is included in theseproliferative factors, and the organic-inorganic hybrid type particlesof then present invention carried thereon with them can be applied to awound part and used for accelerating healing of wound. Further, if ablood coagulating factor, for example, thrombin is carried as abiologically active substance on the above particles in relation tohealing of wound or regardless thereof, it can be used for acceleratinghemostatis in combination with a calcium ion or without combiningtherewith. If the particles carrying (or including) such thrombin areapplied to a wound part in a dry state or a concentrated suspensionstate, it works on fibrinogen present in bleeding blood to form fibrin,which shall bring about so-called hemostatis in situ. Accordingly, acomposition containing such particles is useful as a composition forhemostatis.

[0046] An antiviral agent, an antibacterial agent, an antihistaminicagent, an antitumor agent and a bone inducing agent may be carried asthe other biologically active substance on the above particles accordingto the present invention as long as they can be carried on the aboveparticles.

[0047] According to the present invention, provided is an aqueousdispersion composition for forming the organic-inorganic hybrid typeparticles carried thereon with the biologically active substancecomprising such block copolymer, a calcium ion and a phosphoric acid ionas essential components. The “aqueous dispersion composition” referredto herein or an “aqueous dispersion” referred to later means a solution,a dispersion and a suspension comprising a solvent system whichcomprises water as a principal solvent and which may contain, ifnecessary, a small amount of a water-miscible organic solvent (forexample, methanol, ethanol and acetone) as long as it does not exert anadverse effect in achieving the objects of the present invention. Abuffer which can control the pH to 6.8 to 7.8 is preferably contained inthese solutions. A calcium ion and a phosphoric acid ion each containedin these solutions can originate in the respective correspondingwater-soluble salts. Typically, the former is derived from calciumchloride, and the latter is derived from disodium hydrogenphosphate.

[0048] A content proportion of a calcium ion and a phosphoric acid ionis considerably important in the present invention, and a calcium ionhas to be present in an amount which is excessive as compared with anequivalent required for reacting both to form hydroxyapatite(Ca₁₀(OH)₂(PO₄)₆). To be specific, a proportion of Ca⁺² to PO₄ ³⁻ can be50 to 200: 1 in terms of a mole concentration. When a calcium ion and aphosphoric acid ion are present in such proportion, the block copolymerdescribed above suitably interacts with calcium phosphate(hydroxyapatite) and is bonded, cross-linked or adsorbed.

[0049] Further, Ca⁺² can be present, though shall not be restricted, inan amount of 60 to 300 mM, and PO₄ ³⁻ can be present, though shall notbe restricted, in an amount of 0.4 to 10 mM in the aqueous compositiondescribed above. Such proportions are suited for providing the aqueousdispersion comprising the organic-inorganic hybrid type particlescarrying or including such biologically active substance, including theaqueous composition described above containing the biologically activesubstance according to the present invention. To be specific, the blockcopolymer and the biologically active substance described above suitablyinteract with calcium phosphate (hydroxyapatite) and are bonded oradsorbed. For example, “the particles carried thereon with thebiologically active substance” referred to in the present inventionmeans particles staying in a state in which a part or the whole of thebiologically active substance is included in the inside of the particlesor in which a part or the whole thereof is present on the surface of theparticles.

[0050] On the other hand, the block copolymer can be present, thoughshall not be restricted, in an amount of 10 to 500 μg/ml in the aqueouscomposition described above. The foregoing concentrations of Ca⁺² andPO₄ ³⁻ in the aqueous composition described above and the concentrationof the block copolymer described immediately before are usually suitedfor stably dispersing the organic-inorganic hybrid type particles(containing no biologically active substance) formed in the aboveaqueous composition or the organic-inorganic hybrid type particles(containing the biologically active substance) in the aqueous dispersionin an aqueous solution. However, the aqueous composition or the aqueousdispersion which meets the objects of the present invention can beprovided even if the respective components are used in concentrationsexceeding the concentrations described above. Also, such aqueousdispersion can be converted to a dry form by a conventional method, forexample, a dry freezing method. The composition of such dry form can beconstituted again to a stable aqueous dispersion by adding an aqueousmedium. Further, it can be turned, if necessary, into a formulation ofanother form using another binders as it stays in the dry form.

[0051] The organic-inorganic hybrid type particles described above(containing no biologically active substance) can be used for formingfine or microscopic hydroxyapatite of a submicron order having a uniformparticle diameter. Such particles can be obtained, if necessary, byremoving excess Ca⁺² by dialysis and then freeze-drying.

[0052] The aqueous dispersion according to the present invention whichhas already been partially referred to in the above can be prepared byallowing the biologically active substance to coexist in the aqueouscomposition described above. To be more specific, it can be prepared by,though shall not be restricted, (A) preparing a first aqueous solutioncontaining a biologically active substance, a calcium ion and, ifnecessary, a buffer, (B) preparing independently a second aqueoussolution containing the block copolymer having the structure representedby Formula (I):

PEG-block-poly(carbo)  (I)

[0053] (wherein PEG represents a polyethylene glycol segment, and carborepresents a repetitive unit having a carboxylate ion at a side chain),a phosphoric acid ion and, if necessary, a buffer and (C) mixing thefirst aqueous solution described above with the second aqueous solutionon a condition enough for forming hydroxyapatite. A salt such as sodiumchloride can be contained in the second aqueous solution, and when thebuffer is used, it is preferably selected so that a pH of the finaldispersion can be controlled to 6.8 to 7.8.

[0054] Though shall not be restricted to such production method, theparticles contained in the aqueous suspension according to the presentinvention are particles carried thereon with the biologically activesubstance which are formed from the block copolymer described above, acalcium ion, a phosphoric acid ion and the biologically active substanceand have an average particle diameter of 50 to 600 nm. According to theforegoing production method of the present invention, an aqueousdispersion containing very uniform particles in which an averageparticle diameter has any size of 50 to 600 nm and in which apolydispersity is 0.1 or less can be provided by selecting aconcentration of the block copolymer. It is a matter of course thatparticles having a particle diameter of a several μm order exceeding 600nm can be formed, if necessary, by extending preparing time. Theseaqueous dispersions can be stored for several days to one month on anambient condition (for example, room temperature) without substantiallycausing precipitation or phase separation, and therefore they can beused as a composition for injection as they are or, if necessary, byremoving excess ionic low molecular compounds by dialysis orultrafiltration.

[0055] The biologically active substance which can be carried on suchparticles is, though described above, generally a compound which canshow any useful activity in organisms of animals (particularly humanbeings) and can be polynucleotide (including DNA, mRNA and the like)coding exotic active peptide, polynucleotide coding a function whichpromotes or controls revelation of a specific gene, polynucleotide suchas anti-sense DNA and ribozime and polypeptide (including protein and,as long as the objects are met, oligopeptide). These peptides preferablycontain a polyvalent carboxyl group.

[0056] The organic-inorganic hybrid type particles according to thepresent invention comprise typically, though shall not be restricted,

[0057] 30 to 70% by weight of the block copolymer,

[0058] 25 to 65% by weight of hydroxyapatite and

[0059] 0.1 (preferably 1) to 15% by weight of the biologically activesubstance each based on the whole weight of the above particles.

[0060] The production method for the aqueous dispersion according to thepresent invention can be provided by carrying out the specificembodiment in the presence of a cultured cell, for example, as a methodfor introducing polynucleotide into an animal cell, characterized by:

[0061] (A) adding an aqueous dispersion comprising organic-inorganichybrid type particles carried thereon with a biologically activesubstance to a cultured substance of an animal cell, wherein the aboveparticles are formed from a block copolymer having a structurerepresented by Formula (I):

PEG-block-poly(carbo)  (I)

[0062] (wherein PEG represents a polyethylene glycol segment, and carborepresents a repetitive unit having a carboxylate ion on a side chain),a calcium ion (Ca⁺²), a phosphoric acid ion (PO₄ ³⁻) and the abovebiologically active substance; the above particles have an averageparticle diameter of 50 to 600 nm; and the above biologically activesubstance is selected from the group consisting of poly- oroligonucleotide and poly- or oligopeptide and

[0063] (B) incubating the cultured substance prepared in (A).

[0064] According to such method, the particles can slowly be dissolvedunder a physiologic condition by reducing a concentration of the blockcopolymer in forming the particles described above, and on the otherhand, the particles can stably be maintained under the physiologiccondition by elevating a concentration of the block copolymer, whichmakes it possible as well to control a discharge time of thebiologically active substance in a target part. For example, DNA as thebiologically active substance is introduced into a cell in the form ofthe organic-inorganic hybrid type particles described above, and it canbe delivered to the nucleus of the cell while avoiding decompositioncaused by nuclease in the cell. Such a high efficiency of introducingDNA into a cell can be achieved as well when the composition of thepresent invention is injected into the suited part of an organism, thatis, in a system in vivo.

[0065] As shown above, according to the present invention, the aqueousdispersion comprising the organic-inorganic hybrid type particles whichcan be a carrier for a biologically active substance or the compositionof a dry form can be provided, or the aqueous composition used forproviding the same and the preparing method for them can be provided.

[0066] The present invention shall more specifically be explained belowwhile giving the examples of the specific embodiments of the presentinvention in order to simplify the explanations, but the presentinvention shall not be intended to be restricted to them.

EXAMPLE 1 Experiment for Confirming Action and Effect of Block Copolymer

[0067] This experiment shows that a specific block copolymer iseffective for an inhibition in the formation of precipitates of calciumphosphate (hereinafter abbreviated as CaP) or a controlled formation inparticles having a fixed particle diameter or less.

[0068] <Experimental Method>

[0069] (1) The Following Aqueous Solution was Prepared: Solution A: DNA(16 mer: 70 μg/mL) 1/10 TE buffer (pH 7.6) Ca⁺² 250 mM (using CaCl₂)Solution B: PO₄ ³⁻ 1.5 mM (using Na₂HPO₄) Hepes buffer 50 mM (pH 7.05)NaCl 140 mM poly(aspartic acid) homopolymer (hereinafter abbreviated asPAA) or PEG block-poly (aspartic acid) (hereinafter abbreviated asPEG-PAA; PEG molecular weight: 12000, PAA polymerization degree: 24)

[0070] In the above, PAA is poly(α,β)-DL-aspartic acid, and a compoundhaving a molecular weight of 2000 to 10000 (polymerization degree: 15 to77) (obtained from SIGMA) is used. PEG-PAA is represented by thefollowing formula:

CH₃O(CH₂CH₂O)_(n)—(COCH(CH₂COO⁻)NH)_(x)—(COCH₂CH(COO⁻)NH)_(y)H

[0071] and a compound in which a PEG segment had a molecular weight ofabout 12000 and in which a PAA segment had a polymerization degree (x+y)of 24 was produced and used.

[0072] (2) The solution A was mixed with the solution B at 37° C. totrace a change in a turbidity from a transmission factor of light havinga wavelength of 350 nm.

[0073] <Result>

[0074] The result is shown in FIG. 1. When a homopolymer of PAA wasadded, the transmission factor was suddenly reduced immediately aftermixing the solutions. The degree thereof was dependent on a PAAconcentration, and the higher the polymer concentration was, the morelargely the transmission factor was changed (FIG. 1a). In this case, ifPEG coexisted, the transmission factor was not changed, and it wassuggested that PEG did not interact with precipitate (FIG. 1b). On theother hand, it was shown that the transmission factor was scarcelychanged under the presence of the block copolymer and that precipitatewas inhibited from being formed. It is apparent from these results thatthe copolymer structure is necessary for inhibiting precipitation ofDNA-including CaP.

EXAMPLE 2 Particle Diameter of Composite Particles of DNA and CalciumPhosphate (No. 1)

[0075] The solution A was mixed with the solution B in the same manneras in Example 1 to prepare an aqueous dispersion containing compositeparticles. The dispersion was left standing still at 37° C. for a nightafter mixing, and then the particle diameter was evaluated by dynamiclight scattering (DLS) measurement of the dispersion.

[0076] DSL-7000 manufactured by Ohtsuka Electron Co., Ltd. was used asthe measuring apparatus. A light of argon laser having a wavelength of488 nm was used as an incident light to carry out the measurement at 25°C. A scattered light at an angle 90° to the incident light was detectedto analyze a time dependency in an intensity change thereof by acumulant method, whereby a diffusion coefficient of the particles wasdetermined. The diffusion coefficient thus obtained was converted to theparticle diameter according to the following equation ofStokes-Einstein:

R=kT/(6πηD)

[0077] wherein R=particle diameter, k=Boltzmann's constant, η=viscositycoefficient, D=diffusion coefficient

[0078] <Result>

[0079] The result of measuring the CaP particles by dynamic lightscattering is shown in FIG. 2. It was confirmed by DLS measurement thatthe particles were formed. The particle diameter thereof was 125 nm at aPEG-PAA concentration of 70 μg/ml and decreased as the copolymerconcentration was increased, and it was about 90 nm at 140 μg/ml. Then,the particle diameter was elevated again as the copolymer concentrationwas increased.

[0080] Further, the polydispersity which was an index of a size in aparticle diameter distribution of the particles was determined similarlyby the cumulant method to find that it was 0.1 or less. This value is adeviation of a standardized diffusion coefficient, and when it is 0.1 orless, it is usually regarded as monodispersibility in a colloidalparticle. Refer to FIG. 3.

EXAMPLE 3 Particle Diameter of Composite Particles of Calcium PhosphateContaining No DNA

[0081] The same procedure as in Example 2 was repeated, except that asolution obtained by removing DNA from the solution A prepared inExample 1 was used in producing composite particles. The result thereofis shown in FIG. 4. The particle diameter and the polydispersity arealmost the same as in Example 2.

EXAMPLE 4 Determination of Amount of DNA Introduced (or Included) IntoParticles (No. 1)

[0082] CaP particles were prepared on the same condition as in Example2. The aqueous dispersion thus prepared was subjected to highperformance liquid chromatography (HPLC) on the following conditions toquantitatively determine DNA.

[0083] HPLC condition: column Superose 6HR (room temperature)

[0084] Eluent 1 CaCl₂ 125 mM, 140 mM NaCl, 50 mM HEPES, pH 7.4

[0085] Eluent 2 CaCl₂ 200 mg/L (calcium ion 1.8 mM), NaH₂PO₄.H₂ 0 125mg/L (phosphoric acid ion 0.9 mM) NaCl 6400 mg/L, HEPES 5958 mg/L, pH7.4

[0086] Detection UV 260 nm

[0087] <Result>

[0088] The measuring result of HPLC is shown in FIG. 5. In the case ofDNA alone, a peak originating in DNA was observed in the vicinity of anelution time of 30 minutes. On the other hand, in the case of the CaPsolution, a peak originating in the particles was confirmed togetherwith a peak originating in DNA in the vicinity of an elution time of 12minutes. A proportion of DNA included in the particles was calculatedfrom comparison of a ratio of both.

[0089] Under the conditions of the eluent 1, DNA was decreased from anincluding amount of 45% in a PEG-PAA concentration of 70 μg/mL as theconcentration was increased.

[0090] In the case where the eluent 2 which was close to a physiologiccondition was used, it was suggested that the particles were slowlydissolved at a low polymer concentration. On the other hand, it wassuggested that the particles were stable at a high polymer concentrationeven on a physiologic condition.

[0091] A composition weight ratio of the CaP particles formed can beconverted in the following manner.

[0092] Assuming that calcium is largely excessive in a solutioncontaining 125 mM of calcium and 0.75 mM of phosphoric acid and that allof phosphoric acid molecules are turned into CaP, the composition of CaPis Ca₂(OH)(PO₄)₃, and therefore a CaP weight concentration is 126 μg/ml(Table 1). Further, assuming that all of PRG-PAA added are adsorbed onCaP, the composition ratio shown in TABLE 1 Weight concentration incomponents of CaP particle solution Sample PRG-PAA CaP DNA Total No.(μg/ml) (μg/ml) (μg/ml) (μg/ml) 1 70 126 16 212 2 140 126 11 277 3 210126 7 343 4 280 126 5.3 411

[0093] TABLE 2 Component composition ratio of CaP particles EvaluationPRG-PAA/ CaP/ DNA/ No. weight % weight % weight % 1 33 60 7.4 2 51 463.8 3 61 37 2 4 68 31 1.3

[0094] The eluents 1 and 2 described in the experiments described abovewere used for the following purposes respectively.

[0095] Eluent 1: a calcium concentration of 125 mM in the eluent 1 isthe same as that of the CaP particle solution. The calcium concentrationis largely excessive to that of phosphoric acid on this condition, andit is considered that the CaP particles are not dissolved. In this case,this eluent was used to determine a DNA-including amount in the CaPparticles prepared.

[0096] Eluent 2: a factor exerting an effect on the stability of theparticles on a physiological condition is the concentrations of calciumand phosphoric acid contained in the solution. In this case, a solutioncontaining calcium and phosphoric acid each having a concentration closeto the physiological condition was used as the eluent to evaluate thestability of the particles on the physiological condition.

EXAMPLE 5 Movement of DNA Included in Particles in Cell

[0097] CaP particles were prepared in the same manner as in Example 1.Provided that DNA of 20 mer subjected to fluorescent labeling withRhodamine was used. The aqueous dispersion was left standing still at25° C. for a night and then worked on a cell.

[0098] Experiment of Action on Cell

[0099] A cell strain HuH-7 (originating in human liver cancer, obtainedfrom Riken Gene Bank) was cultivated at 37° C. under 5% CO₂ atmosphereusing a culture medium prepared by adding 10% fetal calf serum to DMEM(Dulbecco's modified eagle medium; obtained from GIBCO BRL).

[0100] The aqueous dispersion was added to the cultured substancedescribed above in a proportion of 1 to 9 of the CaP particle-containingaqueous dispersion to the culture medium in terms of a volume ratio tocultivate the strain for 3 hours, and then the culture medium wasremoved, followed by washing the strain three times with PBS. This wasobserved under a confocal laser microscope LSM510 (manufactured by CarlZeiss Co., Ltd.). Photographs in place of drawings showing the state ofthe treated cells are attached as FIG. 6 to 8.

[0101] CaP/DNA was adsorbed on the surface of the cell on a condition onwhich a precipitate was formed (*precipitate was observed in PEG-PAA 70μg/ml because of a difference in a DNA chain length). On the other hand,it was suggested from a granular fluorescent image that the CaPparticles were introduced into the cell by endocytosis.

[0102] Further, it was suggested that when adding (35 μg/mL, DNA 35μg/mL) poly(methacrylic acid) (molecular weight 9500; obtained fromAldrich Chemical Company Inc.) in forming the particles, DNA islocalized in the nucleus of the cell. DNA is distributed in thecytoplasma only with Rhodamine, and therefore it is considered that DNAis not broken to excess in this case.

EXAMPLE 6 Particle Diameter of Composite Particles of DNA and CalciumPhosphate (No. 2)

[0103] It was shown in Example 2 that a particle diameter of thecomposite particles could be controlled by changing a concentration ofthe copolymer, and it shall be shown in the present example that theabove particle diameter can further be controlled by changing aconcentration of phosphoric acid together with a concentration of thecopolymer.

[0104] <Experiment>

[0105] (1) The following solution was prepared: Solution A: DNA (16 mer:70 μg/mL) 1/10 TE buffer (pH 7.6) Ca⁺² 250 mM (using CaCl₂) Solution B:PO₄ ³⁻ 1.5 mM, 3.0 mM or 6.0 mM (using Na₂HPO₄) Hepes buffer 50 mM (pH7.05) NaCl 140 mM PEG-PAA (PEG molecular weight: 12000, PAApolymerization degree: 24) 140 to 1400 μg/mL

[0106] (2) The solution A was mixed with the solution B each in the sameamount, and the dispersion was left standing still at 37° C. for anight. Then, a particle diameter and a polydispersity of the particleswere determined by dynamic light scattering measurement.

[0107] <Result>

[0108] As shown in FIG. 9(a), a particle diameter of the particlesformed was changed by the concentrations of PEG-PAA and phosphoric acid,and as shown in FIG. 9(b), the uniform particles having a polydispersityof 0.1 or less and a particle diameter of 100 to 300 nm were obtained inany concentration. The marks □, ⋄ and ◯ in FIG. 9(a) and (b) eachcorrespond to the phosphoric acid concentrations of 0.75 mM, 1.5 mM and3.0 mM.

EXAMPLE 7 Determination of Amount of DNA Introduced (or Included) IntoParticles (No. 2)

[0109] Particles prepared according to Example 6 were quantitativelydetermined for an including amount of DNA in the same manner asdescribed in Example 4.

[0110] <Result>

[0111] The measurement result of HPLC is shown in FIG. 10. It isobserved from the drawing that an including amount of DNA tends to go upas a concentration of phosphoric acid is increased. An inclusion rate ofDNA reached 90% in 3.0 mM of phosphoric acid and 540 μg/mL of PEG-PAA.Comparing the results of the eluent 1 with those of the eluent 2, theinclusion rate was observed to be decreased in the eluent 2 in which acalcium concentration and a phosphoric acid concentration were close tothose of a human being. In the drawing, a void shows a result obtainedby using the eluent 1, and a solid mark shows a result obtained by usingthe eluent 2. Numerals in the drawing means the phosphoric acidconcentrations.

EXAMPLE 8 Introduction of DNA-Including Particles Into Cell

[0112] <Experiment>

[0113] 1. Particle Preparing Condition

[0114] (1) The following solution was prepared: Solution A: DNA (16 mer:70 μg/mL) (5′ end was labelled with FITC) 1/10 TE buffer (pH 7.6) Ca⁺²250 mM Solution B: PO₄ ³⁻ 6.0 mM Hepes buffer 50 mM (pH 7.05) NaCl 140mM PEG-PAA (PEG molecular weight: 12000, PAA polymerization degree: 24)1080 μg/mL

[0115] (2) The solutions A and B were mixed in the same amount and leftstanding still at 37° C. for 24 hours.

[0116] 2. Evaluation for Introducing Cell

[0117] An HeLa cell (human cervix cancer cell) was put on a 24-wellplate in a cell density of 2×10⁴/well and cultivated in a DMEM culturemedium (containing 10% FCS) for 24 hours. Then, 50 μL of the particlesor a DNA sample was added to 450 μL of the culture medium (DMEM, 10%FCS). After prescribed time passed, the cell was peeled off bytrypsinization and dispersed in 1 to 2 mL of PBS (cooled with ice untilmeasurement). The cell of about 2×10³ was analyzed by flow cytometry.

[0118] A gate was applied so that the untreated cell having thestrongest fluorescent intensity was contained in a proportion of 1%. Afluorescent intensity of the untreated cell had an average value of 0.5.

[0119] <Result>

[0120] The results are shown in FIG. 11(a), (b) and (c). It can be foundfrom the drawing that DNA alone is scarcely observed to be introduced onthe experimental condition described above. On the other hand, it hasbeen apparent that introduction of DNA into the cell is increased to alarge extent by allowing DNA to be incorporated in the particles andthat nucleic acid is introduced into almost 100% of the cells in 4hours. It can be found as well from a result obtained by plotting anaverage fluorescent intensity of the cell that introduction of DNA intothe cell is promoted to a large extent as compared with DNA alone bymaking use of the particles.

[0121]FIG. 11(a) is a typical histogram (after 24 hours) of theuntreated cell and the cell treated with the CaP particles; (b) shows achange in an amount of introducing DNA into a cell versus time (shown by% of the cell present in a gate area); and (c) shows a change in anamount of introducing DNA into a cell versus time (shown by the averagefluorescent intensity).

EXAMPLE 9 TOXICITY Evaluation Test of Particles

[0122] <Experimental Method>

[0123] 1. Particle Preparing Conditions

[0124] (1) The following solution was prepared: Solution A: DNA (16 mer:70 μg/mL) 1/10 TE buffer (pH 7.6) Ca⁺² 250 mM Solution B: PO₄ ³⁻ 1.5 mMHepes buffer 50 mM (pH 7.05) NaCl 140 mM PEG-PAA (PEG molecular weight:12000, PAA polymerization degree: 24) 140 to 560 μg/mL

[0125] (2) The solutions A and B were mixed in the same amount and leftstanding still at 37° C. for 24 hours.

[0126] 2. Toxicity Test

[0127] An HeLa cell (human cervix cancer cell) was put on a 96-wellplate in a cell density of 5×10³/well and cultivated in a DMEM culturemedium (containing 10% FCS) for 2 days. Then, 10 μL of a particle samplewas added to 90 μL of the culture medium (DMEM, 10% FCS). Aftercultivated for 24 hours, the viable cell number was counted by MTT(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide)assay.

[0128] The MTT assay was carried out in the following manner.

[0129] An MTT (5 mg/mL (PBS)) solution was added in an amount of 10 μLper well of the 96-well plate and left standing still at 37° C. for 2hours. Then, 20% SDS (DMF: water=1:1) was added in an amount of 100 μLper well and left standing still at 37° C. for 24 hours. The absorbanceat 560 nm was measured by a plate leader.

[0130] <Result>

[0131] The result is shown in FIG. 12. FIG. 12 shows percentage of aviable cell number to an untreated cell by a bar graph. According tothis graph, it is apparent that the viable cell number is not decreasedas compared with the untreated cell within an experimental error rangeand that the particles do not show a marked toxicity under the existingexperimental conditions.

EXAMPLE 10 Transfection Experiment

[0132] Plasmid DNA (coded with a luciferase gene) was used as includednucleic acid in the following experiment to investigate an ability ofthe particles for introducing a gene into a cell.

[0133] <Experimental Method>

[0134] 1. Particle Preparation

[0135] (1) The following solution was prepared: Solution A: Plasmid DNA(pGL3-Luc, 5 kbp) (26 to 50 μg/mL) 1/10 TE buffer (pH 7.6) Ca⁺² 250 mMSolution B: PO₄ ³⁻ 1.5 mM Hepes buffer 50 mM (pH 7.05) NaCl 140 mMPEG-PAA (PEG molecular weight: 12000, PAA polymerization degree: 24) 100to 200 μg/mL

[0136] (2) The solutions A and B were mixed in the same amount and leftstanding still at 25° C. for 24 hours.

[0137] 2. Transfection Experiment

[0138] 293T cell (human kidney) was put on a 6-well dish (coated withgelatin) so that 60% confluent was achieved and cultivated in a DMEMculture medium (containing 10% FCS) for 24 hours. After removing theculture medium, the cell was washed with PBS, and a new culture mediumwas added thereto. Then, the sample was added in an amount of 100 μL permL of the culture medium and cultivated for 6 hours. After exchangingthe culture medium, the cell was further cultivated for 24 hours. Theluciferase gene-revealing amount was quantitatively determined by meansof a commercial assay kit.

[0139] <Result>

[0140] The results are shown in FIGS. 13A and B. FIG. 13A shows a resultobtained by examining an influence of a PEG-PAA temperature and thepresence of a blood serum in cultivating for 6 hours to the revelationof a gene. The activity which was higher by 4 to 6 times as comparedwith that in 0 μg/mL was observed in 50 μg/mL of PEG-PAA. Further, it isapparent that the activity revealed is decreased as the PEG-PAAconcentration is increased to 75 and 100 μg/mL.

[0141] The expression amount is reduced on the condition of 50 and 75μg/mL in the presence of a blood serum as compared with in the absencethereof, but the activity which is higher than or equivalent to that in0 μg/mL is obtained.

[0142]FIG. 13B shows a result obtained by examining an influence of theplasmid DNA concentration to the revelation of a gene.

[0143] In a PEG-PAA concentration of 0 μg/mL, a notable difference inrevelation caused by the plasmid DNA concentration was not observed. Onthe other hand, in 50 μg/mL, the revelation went up as the plasmid DNAconcentration was raised.

INDUSTRIAL APPLICABILITY

[0144] The organic-inorganic hybrid particle according to the presentinvention is useful for introducing a biologically active substance (forexample, DNA) into, for example, a cell and expressing it. Accordingly,the present invention is applicable in the medical industry and thepharmaceutical industry.

1. An aqueous dispersion composition for forming organic-inorganichybrid type particles carried thereon with a biologically activesubstance, wherein the particles described above comprise a blockcopolymer having a structure represented by Formula (I):Peg-block-poly(carbo)  (I) (wherein peg represents a polyethylene glycolsegment, and carbo represents a repetitive unit having a carboxylate ionon a side chain), a calcium ion (Ca⁺²) and a phosphoric acid ion (PO₄³⁻) as essential components.
 2. The composition as described in claim 1,wherein the calcium ion is present in an amount which is excessive tothe phosphoric acid ion as compared with an equivalent required forforming hydroxyapatite.
 3. The composition as described in claim 1,wherein carbo is a repetitive unit originating in a carboxylgroup-containing compound selected from the group consisting of asparticacid, glutamic acid, methacrylic acid, acrylic acid andN-acetylhyalobiuronic acid.
 4. The composition as described in claim 1,wherein the above block copolymer is represented by any one of thefollowing Formulas (II-a), (II-b), (III-a) and (III-b):

in the respective formulas, the respective codes each have independentmeanings; A represents a hydrogen atom or a substituted or unsubstitutedalkyl group having up to 12 carbon atoms; L represents a single bond,NH, CO or X(CH₂)_(p)Y, in which X represents OCO, OCONH, NHCO, NHCOO,NHCONH, CONH or COO; Y represents NH or CO; and p represents an integerof 1 to 6; T represents a hydrogen atom, a hydroxyl group or -ZR, inwhich Z represents a single bond, CO, O or NH, and R represents asubstituted or unsubstituted hydrocarbon group having up to 12 carbonatoms; m represents an integer of 4 to 2500; and x+y or z represents aninteger of 5 to 300, provided that a carboxylate ion present can form acarboxyester residue in an amount of up to 50%.
 5. The composition asdescribed in claim 4, wherein m is 12 or more, and x+y or z is 50 orless.
 6. A composition comprising organic-inorganic hybrid typeparticles carried thereon with a biologically active substance, whereinthe particles described above are formed from a block copolymer having astructure represented by Formula (I): PEG-block-poly(carbo)  (I)(wherein PEG represents a polyethylene glycol segment, and carborepresents a repetitive unit having a carboxylate ion on a side chain),a calcium ion (Ca⁺²), a phosphoric acid ion (PO₄ ³⁻) and the abovebiologically active substance, and the above particles have an averageparticle diameter of 50 to 600 nm in an aqueous dispersion.
 7. Thecomposition as described in claim 6, wherein the calcium ion is presentin an amount which is excessive to the phosphoric acid ion as comparedwith an equivalent required for forming hydroxyapatite.
 8. Thecomposition as described in claim 6, wherein carbo is a repetitive unitoriginating in a carboxyl group-containing compound selected from thegroup consisting of aspartic acid, glutamic acid, methacrylic acid,acrylic acid and N-acetylhyalobiuronic acid.
 9. The composition asdescribed in claim 6, wherein the above block copolymer is representedby any one of the following Formulas (II-a), (II-b), (III-a) and(III-b):

in the respective formulas, the respective codes each have independentmeanings; A represents a hydrogen atom or a substituted or unsubstitutedalkyl group having up to 12 carbon atoms; L represents a single bond,NH, CO or X(CH₂)_(p)Y, in which X represents OCO, OCONH, NHCO, NHCOO,NHCONH, CONH or COO; Y represents NH or CO; and p represents an integerof 1 to 6; T represents a hydrogen atom, a hydroxyl group or -ZR, inwhich Z represents a single bond, CO, O or NH, and R represents asubstituted or unsubstituted hydrocarbon group having up to 12 carbonatoms; m represents an integer of 4 to 2500; and x+y or z represents aninteger of 5 to 300, provided that a carboxylate ion present can form acarboxyester residue in an amount of up to 50%.
 10. The composition asdescribed in claim 6, wherein m is 12 or more, and x+y or z is 50 orless.
 11. The composition as described in claim 6, wherein the aboveorganic-inorganic hybrid type particles have any diameter of an averageparticle diameter of 50 to 600 nm and a polydispersity of 0.1 or less inthe aqueous dispersion.
 12. The composition as described in claim 6,wherein the above organic-inorganic hybrid type particles comprise 30 to70% by weight of the block copolymer, 25 to 65% by weight ofhydroxyapatite and 0.1 to 15% by weight of the biologically activesubstance each based on the whole weight of the above particles.
 13. Thecomposition as described in claim 6, wherein the biologically activesubstance is selected from the group consisting of poly- oroligonucleotide and poly- or oligopeptide.
 14. The composition asdescribed in claim 6, wherein the composition comprising theorganic-inorganic hybrid type particles has the form of an aqueousdispersion.
 15. The composition as described in claim 6, wherein thecomposition comprising the organic-inorganic hybrid type particles has afreeze-dried form.
 16. A preparing method for the composition asdescribed in claim 6, comprising: (A) a step of preparing a firstaqueous solution containing a biologically active substance, a calciumion and, if necessary, a buffer, (B) a step of preparing independently asecond aqueous solution containing a block copolymer having a structurerepresented by Formula (I): PEG-block-poly(carbo)  (I) (wherein PEGrepresents a polyethylene glycol segment, and carbo represents arepetitive unit having a carboxylate ion on a side chain), a phosphoricacid ion and, if necessary, a buffer and (C) a step of mixing the firstaqueous solution with the second aqueous solution each described aboveon a condition enough for forming hydroxyapatite.
 17. The preparingmethod as described in claim 16, wherein the biologically activesubstance is selected from the group consisting of poly- oroligonucleotide and poly- or oligopeptide, and the block copolymer isrepresented by any one of the following Formulas (II-a), (II-b), (III-a)and (III-b):

in the respective formulas, the respective codes each have independentmeanings; A represents a hydrogen atom or a substituted or unsubstitutedalkyl group having up to 12 carbon atoms; L represents a single bond,NH, CO or X(CH₂)_(p)Y, in which X represents OCO, OCONH, NHCO, NHCOO,NHCONH, CONH or COO; Y represents NH or CO; and p represents an integerof 1 to 6; T represents a hydrogen atom, a hydroxyl group or -ZR, inwhich Z represents a single bond, CO, O or NH, and R represents asubstituted or unsubstituted hydrocarbon group having up to 12 carbonatoms; m represents an integer of 4 to 2500; and x+y or z represents aninteger of 5 to 300, provided that a carboxylate ion present can form acarboxyester residue in an amount of up to 50%.
 18. A method forintroducing poly- or oligonucleotide into a cell in vitro, comprising:(A) a step of adding an aqueous dispersion comprising organic-inorganichybrid type particles carried thereon with a biologically activesubstance to a cultured substance of an animal cell, wherein the aboveparticles are formed from a block copolymer having a structurerepresented by Formula (I): PEG-block-poly(carbo)  (I) (wherein PEGrepresents a polyethylene glycol segment, and carbo represents arepetitive unit having a carboxylate ion on a side chain), a calcium ion(Ca⁺²), a phosphoric acid ion (PO₄ ³⁻) and the above biologically activesubstance; the above particles have an average particle diameter of 50to 600 nm; and the above biologically active substance is poly- oroligonucleotide and (B) a step of incubating the cultured substanceprepared in (A).
 19. A method for introducing poly- or oligonucleotideinto a cell in vitro comprising: (a) a step of injecting an aqueousdispersion comprising organic-inorganic hybrid type particles carriedthereon with a biologically active substance into a suitable part of ananimal into which the above nucleotide is desired to be injected,wherein the above particles are formed from a block copolymer having astructure represented by Formula (I): PEG-block-poly(carbo)  (I)(wherein PEG represents a polyethylene glycol segment, and carborepresents a repetitive unit having a carboxylate ion on a side chain),a calcium ion (Ca⁺²), a phosphoric acid ion (PO₄ ³⁻) and the abovebiologically active substance; the above particles have an averageparticle diameter of 50 to 600 nm; and the above biologically activesubstance is poly- or oligonucleotide and (b) a step of introducing theabove nucleotide into the above suitable part or a cell present in thevicinity thereof.
 20. A method for producing a dispersion comprisingorganic-inorganic hybrid type particles having an average particlediameter of 50 to 600 nm, comprising a step of carrying out reactionusing a calcium ion and a phosphoric acid ion in an aqueous solution inwhich a block copolymer represented by any one of the following Formulas(II-a), (II-b), (III-a) and (III-b) is present, wherein an excesscalcium ion exceeding an equivalent required for forming hydroxyapatiteis used:

in the respective formulas, the respective codes each have independentmeanings; A represents a hydrogen atom or a substituted or unsubstitutedalkyl group having up to 12 carbon atoms; L represents a single bond,NH, CO or X(CH₂)_(p)Y, in which X represents OCO, OCONH, NHCO, NHCOO,NHCONH, CONH or COO; Y represents NH or CO; and p represents an integerof 1 to 6; T represents a hydrogen atom, a hydroxyl group or -ZR, inwhich Z represents a single bond, CO, O or NH, and R represents asubstituted or unsubstituted hydrocarbon group having up to 12 carbonatoms; m represents an integer of 4 to 2500; and x+y or z represents aninteger of 5 to 300, provided that a carboxylate ion present can form acarboxyester residue in an amount of up to 50%.